Figure 4.
Proteasome Inhibition Increases Intra-neuronal ASPD Accumulation
(A) Primary rat hippocampal neuronal cultures, with or without AAV-APPswe transduction, were treated with 75 nM MG132 for 24 h. The cultures were triple stained with antibodies against N-terAβ, MAP2, and DAPI on the right side, or against MAP2, GFAP, and ASPD (rpASD1) on the left side. Representative images are shown. Scale bar, 50 μm. In this work, ASPD were detected by immunohistochemical staining with ASPD-specific rpASD1 antibody or mASD3 antibody (Noguchi et al., 2009), both of which have been used to detect ASPD in immunopathological studies of human brains (Noguchi et al., 2009, Ohnishi et al., 2015).
(B) Primary rat hippocampal neuronal cultures, with or without AAV-APPswe transduction, were treated with 75 nM MG132 for the indicated time. The cultures were double stained with anti-ASPD rpASD1 antibody and DAPI. An enlarged view of the field enclosed by a hatched line is shown immediately on the right. Representative images are shown. Scale bar, 50 μm. The number of ASPD and MAP2 double-positive cells was counted by using a GE Healthcare Life Sciences “IN Cell Analyzer” system and is shown as “ASPD + neuron” in the inset upper panel. Then the total amount of rpASD1-immunostained ASPD that was detected in MAP2-positive cells was determined by using an IN Cell Analyzer and was divided by the number of ASPD and MAP2 double-positive cells, which is shown as “ASPD level/neuron” in the inset lower panel (mean ± SD, n = 3; Scheffé post hoc test; ***p < 0.0001, **p = 0.0006, *p = 0.0176).